CD1d-restricted T cells (or "NKT" cells) recognize lipid and gtycolipid antigens, and have functions that can impact a variety of immunological processes. They can contribute to Th1 responses that mediate tumor rejection and defense against a variety of microbial infections, and they also appear to play an important role in preventing autoimmune disease. How these contrasting functions are regulated remains unclear, but an important feature is likely to be the nature of the antigenic stimulus. The strength of the TCR signal encountered by an NKT cell may critically affect the type of functional response that is elicited, and different antigens may vary in signalling strength. For example, administration of the synthetic glycolipid (-GaICer powerfully stimulates CD1d-restricted T cells, resulting rapidly in potent secretion of both Th1 and Th2 cytokines, and other effector functions. In contrast, recognition of self antigens produces weaker responses, which in the absence of inflammatory co-stimulation may result in NKT cell functions that promote peripheral tolerance. These studies will investigate how the TCR structure of CD1d-restricted T cells determines antigen recognition and reactivity. The approach utilizes CD1d-restricted T cell clones that have novel "non-cannonical" TCRs, and which differ from V(24-invariant T cells in distinguishing between two closely related lipids. These exceptional CD1d-restricted T cells provide a unique opportunity to correlate TCR structure with functional recognition of lipid antigens. The specific aims are : i) investigate the relationship between CD1d-restricted T cells responses to different lipids and TCR sequence; ii) identify and test TCR structural features that determine antigen and CD1d recognition; iii) investigate how T cell responses to different lipid antigens relate to TCR affinity for the antigen, immunological synapse formation, and TCR signal transduction. Completion of these aims will provide a better understanding of how the antigen specificity of NKT cells is determined, and how recognition of different antigens relates to functional activation. Understanding how to stimulate these multi-faceted T cells to achieve specifically the desired response will be critical to the ability to exploit their varied functions for uses such as providing short-term immunostimulation in the event of a bio-terrorist attack, promoting effective anti-tumor responses, and enhancing peripheral tolerance mechanisms to combat autoimmune disease.